ABSTRACT:
A detailed molecular model for alkali-denatured duplex
circular DNA (“Form IV”) is proposed. The illustrative biological example used is
the replicative form of fx174, a 5 kb duplex
circular chromosome. The model explains all of Form IV’s known and peculiar features.
In a sedimentation coefficient vs. pH titration, Form IV begins to appear at pH
12.3, at which point it can be persuasively argued that no further supertwists can
be added to the already-highly-supertwisted chromosome. Therefore a new structure
must appear. The sedimentation coefficient s then undergoes a massive, but
initially reversible increase as the pH is raised further, culminating at pH 12.8
with a 250% increase. This degree of compactness can only be explained by a 4-stranded
tetraplex structure, consisting of a pair of duplexes whose base pairs are mutually
intercalated. Above pH 12.8, the structural changes become irreversible, suggesting a further conformational
change. It is proposed that this involves an axial rotation of the component duplex
strands, so that the bases now stack on the outside, and the phosphate groups lie
in the core, where they bond ionically by means of salt bridges. When the irreversibly
denatured compact structure is neutralized at moderate-to-high salt concentrations,
a third novel structure appears, which has a sedimentation coefficient midway between
the native 21 s and the
denatured 50 s. It is proposed that this is a hybrid structure; part tetraplex,
part duplex. To return to a fully-duplex form, it is necessary to both neutralize
the solution, and also to greatly reduce the ionic strength, i.e., to the range 0.001-0.01 M. Since the DNA, under those conditions,
cannot possibly have normal complementary base-pairing, the duplex structure must
either be tautomerically base-paired, or else stabilized solely by base-stacking,
with no base-pairing at all.